Preclinical Evaluation of Recombinant Microbial Glycoside Hydrolases in the Prevention of Experimental Invasive Aspergillosis.

Aspergillus fumigatus is a ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections. IMPORTANCE The biofilm-forming mold Aspergillus fumigatus is a common causative agent of invasive fungal airway disease in patients with a compromised immune system or chronic airway disease. Treatment of A. fumigatus infection is limited by the few available antifungals to which fungal resistance is becoming increasingly common. The high mortality rate of A. fumigatus-related infection reflects a need for the development of novel therapeutic strategies. The fungal biofilm matrix is in part composed of the adhesive exopolysaccharide galactosaminogalactan, against which antifungals are less effective. Previously, we demonstrated antibiofilm activity with recombinant forms of the glycoside hydrolase enzymes that are involved in galactosaminogalactan biosynthesis. In this study, prophylaxis with glycoside hydrolases alone or in combination with the antifungal posaconazole in a mouse model of experimental aspergillosis improved outcomes. This study offers insight into the therapeutic potential of combining biofilm disruptive agents to leverage the activity of currently available antifungals.

Fluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - A cautionary disclosure.

Fluorination of metabolic hotspots in a molecule is a common medicinal chemistry strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.

Convergent synthesis of new N -substituted 2-{[5-(1H -indol-3-ylmethyl)-1, 3, 4-oxadiazol-2-yl]sulfanyl}acetamides as suitable therapeutic agents

abstract A series of N-substituted 2-{[5-(1H-indol-3-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl}acetamides (8a-w) was synthesized in three steps. The first step involved the sequential conversion of 2-(1H-indol-3-yl)acetic acid (1) to ester (2) followed by hydrazide (3) formation and finally cyclization in the presence of CS2 and alcoholic KOH yielded 5-(1H-indole-3-yl-methyl)-1,3,4-oxadiazole-2-thiol (4). In the second step, aryl/aralkyl amines (5a-w) were reacted with 2-bromoacetyl bromide (6) in basic medium to yield 2-bromo-N-substituted acetamides (7a-w). In the third step, these electrophiles (7a-w) were reacted with 4 to afford the target compounds (8a-w). Structural elucidation of all the synthesized derivatives was done by 1H-NMR, IR and EI-MS spectral techniques. Moreover, they were screened for antibacterial and hemolytic activity. Enzyme inhibition activity was well supported by molecular docking results, for example, compound 8q exhibited better inhibitory potential against α-glucosidase, while 8g and 8b exhibited comparatively better inhibition against butyrylcholinesterase and lipoxygenase, respectively. Similarly, compounds 8b and 8c showed very good antibacterial activity against Salmonella typhi, which was very close to that of ciprofloxacin, a standard antibiotic used in this study. 8c and 8l also showed very good antibacterial activity against Staphylococcus aureus as well. Almost all compounds showed very slight hemolytic activity, where 8p exhibited the least. Therefore, the molecules synthesized may have utility as suitable therapeutic agents.

resumo Uma série de acetamidas 2-{[5-(1H-indol-3-ilmetil)-1,3,4-oxadiazol-2-il]sulfanila} N-substituídas (8a-w) foi sintetizada em três fases. A primeira etapa envolveu a conversão sequencial de ácido 2-(1H-indol-3-il)acético (1) a éster (2), seguido por hidrazida (3) e, finalmente, a e ciclização na presença de CS2 e KOH alcoólico produziu 5-(1H-indol-3-il- metil)-1,3,4-oxadiazole-2-tiol (4). Na segunda etapa, aminas arílicas/aralquílicas(5a-w) reagiram com brometo de 2-bromoacetila (6​​), em meio básico, para se obter acetamidas 2-bromo-N-substituídas (7a-w). Na terceira etapa, estes eletrófilos (7a- w) reagiram com 4, para se obter os compostos alvo (8a-w). A elucidação estrutural de todos os derivados sintetizados foi realizada por 1H-NMR, IR e técnicas de espectrometria de EI-MS. Além disso, eles foram submetidos a triagem de atividade antibacteriana e hemolítica. Análise da inibição enzimática foi bem apoiada pelos resultados de docking molecular. Por exemplo, o composto 8q exibiu melhor potencial inibitório contra α-glicosidase, e os compostos 8g e 8b exibiram, comparativamente, melhor inibição contra butirilcolinesterase (BChE) elipoxigenase (LOX), respectivamente. Do mesmo modo os compostos 8b e 8c mostraram excelente potencial antibacteriano contra SalmonellaTyphi, semelhante ao do ciprofloxacino, antibiótico padrão usado neste estudo. Os compostos 8c e 8l também mostraram excelente potencial antibacteriano contra Staphylococcus aureus . Quase todos os compostos mostraram pequena atividade hemolítica, sendo que o composto 8p apresentou menor atividade. Assim, as moléculas sintetizadas podem ter a sua utilidade como agentes terapêuticos adequados.

Functional and structural studies of pullulanase from Anoxybacillus sp. LM18-11.

Pullulanase is a debranching enzyme that specifically hydrolyzes the α-1,6 glycosidic linkage of α-glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18-11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and Km for pullulan of PulA are 750 U mg(-1) and 16.4 µmol L(-1), respectively. PulA has a half-life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75-2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third ß-strand and the third α-helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate-binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain.

Hydration of vinyl ether groups by unsaturated glycoside hydrolases and their role in bacterial pathogenesis

Many pathogenic microorganisms invade mammalian and/or plant cells by producing polysaccharide-degrading enzymes (lyases and hydrolases). Mammalian glycosaminoglycans and plant pectins that form part of the cell surface matrix are typical targets for these microbial enzymes. Unsaturated glycoside hydrolase catalyzes the hydrolytic release of an unsaturated uronic acid from oligosaccharides, which are produced through the reaction of matrix-degrading polysaccharide lyase. This enzymatic ability suggests that unsaturated glycoside hydrolases function as virulence factors in microbial infection. This review focuses on the molecular identification, bacterial distribution, and structure/function relationships of these enzymes. In contrast to general glycoside hydrolases, in which the catalytic mechanism involves the retention or inversion of an anomeric configuration, unsaturated glycoside hydrolases uniquely trigger the hydrolysis of vinyl ether groups in unsaturated saccharides but not of their glycosidic bonds (AU)

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Significance of immune response to enzyme-replacement therapy for patients with a lysosomal storage disorder.

Lysosomal storage disorders are collectively important because they cause significant morbidity and mortality. Patients can present with severe symptoms that include somatic tissue and bone pathology, developmental delay and neurological impairment. Enzyme-replacement therapy has been developed as a treatment strategy for patients with a lysosomal storage disorder, and for many of these disorders this treatment is either in clinical trial or clinical practice. One major complication arising from enzyme infusion into patients with a lysosomal storage disorder is an immune response to the replacement protein. From clinical trials, it is clear that there is considerable variability in the level of immune response to enzyme-replacement therapy, dependent upon the replacement protein being infused and the individual patient. Hypersensitivity reactions, neutralizing antibodies to the replacement protein and altered enzyme targeting or turnover are potential concerns for patients exhibiting an immune response to enzyme-replacement therapy. The relative occurrence and significance of these issues have been appraised.

The effects of a pre-exercise feeding with or without fungal carbohydrases (Carbogen) on blood parameters and exercise performance in elite cyclists: a preliminary study.

The effect of fungal carbohydrases (Carbogen[C]) consumed with a meal replacement bar (MBR) on glucose metabolism and exercise performance was determined in 5 male competitive cyclists. After a 12-hour fast, subjects performed two 60-min cycling bouts at 80% VO2max followed by a time-to-exhaustion (TE) ride at 100% VO2max. One hour prior to each cycling bout, subjects ingested a MRB + 160-mg C or 160-mg CaCO3 placebo (PL) in a double-blind, counterbalanced fashion. Blood was drawn for determination of glucose, insulin, and lactate at: fasting, 1 hour post-feeding, minutes 30 and 60 of exercise, and after TE. Two-way ANOVA revealed a significant (p <.05) treatment and time effect for glucose, with C being higher than PL. Interaction effects were observed for insulin and lactate. An increase in TE (min) at 100% VO2max was observed in the C versus PL trial (6.3 3.4 vs. 4.4 2.9, p <.001). A MRB+C may benefit cyclists due to increased BG and improved exercise performance.

Mannose-receptor-mediated clearance of lysosomal alpha-mannosidase in scavenger endothelium of cod endocardium.

Mannose-receptor-mediated clearance of circulating glycoproteins was studied in Atlantic cod (Gadus morhua). Distribution studies with radioiodinated and fluorescently labelled ligands showed that cod liver lysosomal alpha-mannosidase and yeast invertase were rapidly eliminated from blood via a mannose specific pathway in liver parenchymal cells and endocardial endothelial cells of atrium and ventricle. Asialo-orosomucoid, a galactose-terminated glycoprotein, was cleared by liver only. In vitro studies were performed with primary cultures of atrial-endocardial endothelial cells (AEC), incubated at 12 degrees C in a serum free medium. Cod AEC endocytosed mannose-terminated glycoproteins (125I-alpha-mannosidase, 125I-invertase, 125I-mannan, 125I-ovalbumin and unlabelled lysosomal alpha-mannosidase), whereas 125I-asialo-orosomucoid was not recognised. Uptake of radiolabelled mannose-terminated ligands was inhibited 80-100% in the presence of excess amounts of mannan, invertase, D-mannose, L-fucose or EGTA. Our results suggest that the cod endocardial endothelial cells express a specific Ca(2+)-dependent mannose receptor, analogous to the mannose receptor on mammalian macrophages and liver sinusoidal endothelial cells.

Cell surface expression of polysialic acid on NCAM is a prerequisite for activity-dependent morphological neuronal and glial plasticity.

Polysialic acid (PSA) on the extracellular domain of the neural cell adhesion molecule (NCAM) reduces cell adhesion and is considered an important regulator of cell surface interactions. The hypothalamo-neurohypophysial system (HNS), whose glia, neurons, and synapses undergo striking, reversible morphological changes in response to physiological stimulation, expresses high levels of PSA-NCAM throughout life. Light and electron microscopic immunocytochemistry in normal rats and rats in which cell transport was blocked with colchicine showed that PSA-NCAM is expressed in both HNS neurons and glia, particularly at the level of astrocytic processes that envelop neuronal profiles and can undergo remodeling. Moreover, we confirmed that the overall levels of PSA-NCAM were not greatly altered by stimulation (lactation and chronic salt ingestion). Nevertheless, PSA is essential to morphological plasticity. Using comparative ultrastructural analysis, we found that, after specific enzymatic removal of PSA from NCAM by microinjection of endoneuraminidase close to the hypothalamic magnocellular nuclei in vivo, there was no apparent withdrawal of astrocytic processes nor any increase in synaptic contacts normally induced by lactation and dehydration. Our observations demonstrate, therefore, that expression of PSA on cell surfaces in the adult HNS is indispensable to its capacity for activity-dependent morphological neuronal-glial and synaptic plasticity. The carbohydrate PSA on NCAM can thus be considered a necessary permissive factor to allow neuronal and glial remodeling to occur whenever the proper inductive stimulus intervenes.

Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles.

Electron microscopy was used to evaluate the function and formation of dense core lysosomes. Lysosomes were preloaded with bovine serum albumin (BSA)-gold conjugates by fluid phase endocytosis using a pulse-chase protocol. The gold particles present in dense core lysosomes and late endosomes were flocculated, consistent with proteolytic degradation of the BSA. A second pulse of BSA-gold also accumulated in the pre-loaded dense core lysosomes at 37 degrees C, but accumulation was reversibly blocked by incubation at 20 degrees C. Time course experiments indicated that mixing of the two BSA-gold conjugates initially occurred upon fusion of mannose 6-phosphate receptor-positive/lysosomal glycoprotein-positive late endosomes with dense core lysosomes. Treatment for 5 hours with wortmannin, a phosphatidyl inositide 3-kinase inhibitor, caused a reduction in number of dense core lysosomes preloaded with BSA-gold and prevented a second pulse of BSA-gold accumulating in them. After wortmannin treatment the two BSA-gold conjugates were mixed in swollen late endosomal structures. Incubation of NRK cells with 0.03 M sucrose resulted in the formation of swollen sucrosomes which were morphologically distinct from preloaded dense core lysosomes and were identified as late endosomes and hybrid endosome-lysosome structures. Subsequent endocytosis of invertase resulted in digestion of the sucrose and re-formation of dense core lysosomes. These observations suggest that dense core lysosomes are biologically active storage granules of lysosomal proteases which can fuse with late endosomes and be re-formed from the resultant hybrid organelles prior to subsequent cycles of fusion and re-formation.

[Enzymatic kinetics and mid-infrared spectroscopy combined with multidimensional data analysis]. / Cinétique enzymatique et spectroscopie moyen infrarouge combinée avec l'analyse multidimensionnelle de données.

An alternate method for enzyme study is proposed. This technique allows enzymatic reactions by a one step assay, and visualisation of variations in FTIR spectral data of substrate during the reaction. Hydrolysis of sucrose by beta-fructosidase is carried out as an example.

Adsorption mode of exo- and endo-cellulases from Irpex lacteus (Polyporus tulipiferae) on cellulose with different crystallinities.

The adsorption mode of two highly purified cellulases, exo- and endo-type cellulases, from Irpex lacteus (Polyporus tulipiferae) was investigated by using pure cellulosic materials with different crystallinity as substrates. Adsorption of the two enzymes on the substrates was found to fit the Langmuir-type adsorption isotherm. Maximum amount of adsorbed enzyme obtained from the Langmuir plots showed an inverse correlation to the crystallinity of the substrate with both enzymes, and this value of endo-type cellulase was less dependent on the degree of crystallinity of substrates than that of exo-type cellulase, whose isotherms reached saturation in the range of low enzyme concentrations. The two enzymes showed relatively high affinities for all the substrates and their affinities increased with increasing crystallinity, but this tendency was less marked with endo-type cellulase than with exo-type one. In addition, large negative values of free energy change were observed on the adsorption of both enzymes, and the values became more negative with increasing crystallinity. Consequently, both cellulases showed high adsorption on crystalline cellulose and the adsorption process became smoother with increasing crystallinity. The adsorption of the two types of cellulases was endothermic with an increase in entropy, especially for amorphous cellulose, suggesting the occurrence of water release from the substrates during enzyme adsorption. In addition, the changes in thermodynamic parameters (delta H, delta S, and delta G) in adsorption of exo-type cellulase were larger than in that of endo-type enzyme.

[Distribution studies of alpha-L-arabinofuranosidase already unstable at pH 7.0 (37 degrees C) in tumor-bearing mice in connection with its possible use to enhance the selectivity of the chemotherapy of malignant tumors]. / Untersuchungen zur Verteilung einer bereits bei pH 7.0 (37 Grad C) instabilen alpha-L-Arabinofuranosidase in tumortragenden Mäusen im Zusammenhang mit ihrer möglichen Anwendung zur Erhöhung der Selektivität der Chemotherapie maligner Tumoren.

Graffi et al. (1-3) had proposed the use of exogenous enzymes to toxify inactive transport forms of cancerostatic substances. For this purpose, the pH difference between normal tissues and the tumor was to be exploited, which can be essentially increased by the application of glucose and inorganic phosphate (5-7). Earlier studies using alpha-L-arabinofuranosidase obtained from Aspergillus niger have shown that the selectivity of tumor chemotherapy can be increased in this way (4). The alpha-L-arabinofuranosidases known to date are stabile in a wide pH range (9). However, in some moulds we found pH-labile enzymes of this kind that become irreversibly inactivated in the weakly alkaline or neutral pH range (10, 11). Studies on the distribution of the activity of a pH-labile alpha-L-arabinofuranosidase from Glomerella myabana in tumor-bearing mice have shown that this enzyme is rapidly eliminated from the organism, in contrast to the pH-stable alpha-L-arabinofuranosidase from A. niger. Apart from its excretion via kidney and liver, of importance is the inactivation of the enzyme in the normal tissues. The additional application of glucose strongly increased the activity of this enzyme both in the tumor and in normal tissues (12). By injecting alkaline solutions, stronger inactivation in normal tissues than in the tumor was achieved (13). In the present paper, distribution of an alpha-L-arabinofuranosidase from Fusarium species I 50 (11), inactive already at pH 7.0 (37 degrees C), was studied in tumor-bearing mice. The activity of this enzyme could be enriched under various conditions in the tumor, and especially favorable proved to be the additional application of a combination of glucose and inorganic phosphate. Under these conditions, a higher activity than in the tumor was demonstrable only in the kidney, which can possibly be eliminated in larger experimental animals by diuretics or an appropriate alkaline administration. The investigations have shown that the pH-labile alpha-L-arabinofuranosidases, especially those of Fusarium sp., due to their pharmacokinetic behavior are better suited for use in our therapy concept than the hitherto employed enzyme from A. niger. More recently, Tietze (16) has proposed a similar therapy concept, in which also the glucose-increased pH difference between tumor and normal tissue using tumor-own enzymes, exogenous enzymes as well as transport forms of cancerostatic agents spontaneously hydrolysing under weakly acidic pH conditions is to be exploited.

[Experiments on the modification of the distribution of the activity of various alpha-L-arabinofuranosidases following i.v. application in tumor-bearing mice]. / Versuche zur Beeinflussung der Verteilung der Aktivität verschiedener alpha-L-Arabinofuranosidasen nach i.v. Applikation in tumortragende Mäuse.

Graffi et al. have proposed the use of exogenous enzymes for selective cleavage of inactive transport forms of cancerostatic substances in tumor tissue. Enzymes appropriate for this purpose should, if possible, show also a certain enrichment in the tumor tissue. In studies on the pH-labile alpha-L-arabinofuranosidase from G. myabena in tumor-bearing mice, under defined conditions in the tumors there could be demonstrated a higher concentration of the active form of the enzyme than in most of the normal tissues. However, the enzyme activity is eliminated from the organism in a relatively short time through excretion and inactivation. The application of glucose led to a strong increase of activity of the enzyme injected, both in the tumors and in the normal tissues. It has been shown in the present investigations that this increase in the enzyme activity can be curtailed more strongly in normal tissue by alkali application than in the tumors. In this way, a distribution of the enzyme activity favorable for therapy experiments is obtained. Only in the kidney and urine has a higher activity of the applied enzyme been measured than in the tumors. In the second part of this work it has been attempted to achieve accumulation of activity of the pH-stable alpha-L-arabinofuranosidase from A. niger through application of angiotensin, but no positive results have been reached under the various experimental conditions used.

[Pharmacokinetic studies of the activity of a pH-labile alpha-L-arabinofuranosidase following i.v. injection into tumor-bearing mice]. / Pharmakokinetische Untersuchungen der Aktivität einer pH-labilen alpha-L-Arabinofuranosidase nach i.v.-Injektion in tumortragende Mäuse.

In order to increase the selectivity of tumor chemotherapy, Graffi et al. have proposed the application of xenogenic enzymes, which are able to split transport forms of carcinostatics under the pH-conditions in the tumor more vigorously than in the normal tissues. This paper describes the distribution within the body and elimination of the activity of the pH-labile alpha-L-arabinofuranosidase from G. myabena compared with the pH-stable enzyme from A. niger, using tumor bearing mice. In vitro, the pH labile arabinosidase was irreversibly inactivated within a few minutes at pH 7.4 and 37 degrees C; however at pH 6.5 it remained active even after several hours. After injection, this enzyme activity was eliminated from the organism by excretion and inactivation within a few hours. Hereby a relatively favourable distribution of the enzyme activity for therapeutic application was reached after 60 minutes. At this time higher activity than in the tumor was measured only in the kidney. The application of glucose led to a strong increase of the enzyme activity in both tumor and normal tissues. This effect was also seen in tumor free mice. In further experiments it will be tried to find out conditions which reduce the glucose induced acidosis. The activity and distribution of the pH-stabile enzyme from A. niger were not influenced by glucose application.